Hepg2 cell

Nrf1α^−/− cells were established by TALENs-led genome editing with HepG2 cells [26 ], whereas Nrf2^−/− cells were constructed by CRISPR/Cas9-editing system with HepG2 cells [29 ]. These cell lines expressing Nrf1α and Nrf2, as well as an empty control, were established by using the Flp-In™ TREx™-293 system with HepG2 cells (Invitrogen) [30 ]. These cell lines were maintained in DMEM supplemented with 5 mM glutamine, 10% (v/v) fetal bovine serum (FBS), and 100 units/mL of either penicillin or streptomycin, in the 37 °C incubators with 5% CO₂. In addition, some cell lines were transfected for 8 h with the indicated constructs mixed with the Lipofectamine®3000 agent in the Opti-MEM (gibca, Waltham, MA, USA). The cells were then allowed for recovery from transfection in a fresh complete medium for 24 h before the other experiments were conducted.

Human hepatoma HepG2 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM)-F12 (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% (v/v) fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C under 5% CO₂. HepG2 cells were co-transfected with HBc mutant plasmids and wild-type (WT) pHBV1.2. 1–183 flag and pcDNA3.1 were also transfected into HepG2 cells to establish control groups. All transfections were conducted using FuGENE HD transfection reagent (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocols. The day prior to transfection, 5×10⁵ cells were seeded into a 6-well plate and cultured at 37°C. After 20 h, 6 µl FuGENE HD reagent and 2 µg HBc mutant plasmid or different amounts of HBc mutant plasmids (0.2–3.2 µg) together with 1 µg pHBV1.2 were used for transfection. According to preliminary experiments using a pCMV-GFP plasmid, ~35% of target HepG2 cells were successfully transfected. Five days post-transfection, cells were harvested for further analysis.

HepG2 cells (American Type Culture Collection, Manassas, VA) were used and maintained in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL amphotericin B, as previously reported.³¹ (link) The pCMV6-based mammalian expression vectors, empty (used as a control) and encoding HIF-2α (OriGene), were used to generate and select HepG2 cells stably overexpressing HIF-2α.³¹ (link) HepG2 cells were seeded and then transfected 24 hours later with 10 μg of each vector using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). HIF-2α expression of the generated stable transfectants was carefully characterized, after which the cell lines carrying the empty vector (H/V6) and overexpressing HIF-2α (H/2α) then were used for the experiment described.
H/2α and control cells containing the empty pCMV6 vector (H/V6) were grown in Dulbecco’s modified Eagle medium under normoxic conditions to obtain the desired subconfluence level (65%–70%).